A novel type of vaccines based on plasmids expressing microbial antigens upon transfection of mammalian cells was developed recently. Intramuscular inoculation of mice with this type of 'DNA vaccines' was shown to induce an immune response including antibodies, cytolytic T (Tc) and T helper (T/H) cells and thus protection against challenge with the pathogen. The immune response to the DNA vaccines was of unexpected duration, suggestive of persistence of vector-encoded antigens. The goal of this application is to use the well-established murine rabies model to increase our knowledge of the molecular and immunological basis of DNA vaccination. Emphasis is given to unravel the basis for the persistence of the immune response to DNA vaccines and its immunological consequences. For this, vectors based either on different forms of viral proteins expressed by vectors and, in addition, co-transfection with immunologically relevant molecules will be used. The efficacy of DNA vaccines is presumably correlated with the rate of transcription of vector-encoded microbial genes that determines the level of expression of foreign proteins. To test for the effect of transcription rates the immune response to vectors that express the rabies virus glycoprotein (G protein) under the control of different viral promoters will be compared. The efficacy of DNA vaccines to induce immune responses will depend on the type of transfected and thus antigen-expressing cells and their ability to present the antigen in an immunogenic fashion to the immune system. To investigate the role of different cell types, i.e., muscle cells vs. MHC class II+ antigen-presenting cells that might be transfected upon vector immunization, tissue-specific promoters (MHC class II Ea promoter and the muscle creatine kinase enhancer/promoter) will be used in DNA vaccine constructs. The magnitude of the immune response to the DNA vaccine will depend on characteristics of the foreign antigen such as its cellular localization that might influence antigen- presentation. This will be tested by comparing the immune response to a truncated viral G protein, that, lacking the transmembrane domain, is secreted from transfected cells, wild-type G protein expressed on the cell-surface and a differently truncated G protein that due to removal of the signal sequence remains in the cytoplasma of transfected cells. The type of immune response (T/c vs. T/H cells) will depend on epitopes expressed on vector-encoded proteins. The interplay between different immune effector mechanisms will be tested using vectors expressing internal proteins of rabies virus, i.e., the nucleoprotein that, as presented by rabies virus, is the dominant target antigen for T/H cells but fails to induce T/c cells and the nominal phosphoprotein that induces an excellent T/c cell response but only a marginal T/H cell response. As an additional approach to potentiate DNA vaccines G protein-expressing vectors will be co-injected with vector expressing interleukins (IL) -2, IL-4 and IL-12 known to have extensive effects on activation of immune responses. The last set of experiments is designed to address the basis for the persistence of the immune response to a DNA vaccine that is highly suggestive of long-lasting expression of antigen. This could be caused by T cell ignorance as will be addressed by co-transfection with an IFN/gamma expressing vector, or by T cell anergy as will be tested by co-transfection with a vector expressing a co-stimulatory signal, i.e., B7.